Couette Flow Linear Dichroism Spectroscopy

Linear Dichroism (LD) spectroscopy uses linearly polarized light to measure how a molecule orients itself with respect to a reference axis or, more precisely, how the transition moment of a chromophore in the molecule is oriented. This provides insight into how, e.g., molecules assemble into macromolecules and their orientation with respect to each other. Prerequisites for LD is a medium in which the molecules orient in a preferred direction and that the molecules under investigation are long enough to allow orientation. For long biomolecules such as RNA/DNA or fibrils of proteins in solution, Couette flow is an efficient way of creating a flow-induced orientation of the molecules, while recirculating a small volume of sample. In this chapter, we present the methodology behind LD spectroscopy including best practice for sample handling and data collection, as well as the interpretation of LD spectra including examples of RNA, DNA, proteins, and their complexes.